PROJECT SUMMARY Cellular health depends upon the proper folding and functioning of proteins. Protein misfolding is associated with an ever-expanding list of pathologies, including Alzheimer?s disease, amyotrophic lateral sclerosis, mad cow disease and other amyloidoses. It is likely that misfolding represents an underlying cause of many non- amyloidogenic genetic disorders as well, since destabilization of protein structure via missense mutation can give rise to alternative conformations more prone to degradation or aggregation. Deciphering how protein sequences predispose a structure to misfold will allow for better prediction of mutational effects on cellular health and more generally elucidate the physical constraints on protein evolution. The long-term objectives for the proposed research is to understand the relationship between proteins? biophysical properties and their impact on organismal health and fitness. The projects described here are inherently interdisciplinary, bridging the fields of protein biophysics and evolutionary biology and including experiments that span scales of complexity from in vitro to in vivo. They propose using b-lactamases, a family of enzymes involved in antibiotic resistance, to explore fundamental questions about (Aim 1) how a protein?s sequence specifies its energy landscape and (Aim 2) which features of the landscape are subject to evolutionary selective pressures. Aim 1 focuses on characterizing partially-folded conformations in two structurally-related b- lactamase homologs, TEM and CTX-M, and determining how they are encoded by sequence using spectroscopic (circular dichroism, fluorescence, NMR) and protein engineering techniques. The goal of Aim 2 is to examine the functional and fitness impacts of these partially-folded conformations and explore other potentially relevant energetic features such as kinetic and thermodynamic stabilities using quantitative metrics such as proteolytic susceptibility and relative fitness. Successful completion of these projects will not only shed light on the specifics of b-lactamase evolution, potentially enabling prediction of novel resistance mutations, but also provide paradigmatic molecular explanations for why particular missense mutations are adaptive, deleterious or neutral.